Iris is a winter hardy, herbaceous perennial consisting of approximately 300 species, many of which are popular ornamentals in the temperate regions of the Northern Hemisphere. Most horticulturally important irises are bearded species and their hybrids are derived from species native to the Near East and Europe (Kohlein, Iris, Timber Press, Portland, Oreg., 1987). In addition to their ornamental value, certain species, such as Iris pallida Lam. and Iris germanica L., contain an essential oil composed partly of irones that can be extracted from rhizomes (Jxc3xa9han et al., Plant Cell Rpt., 13:671-675, 1994; Kohlein, Iris, Timber Press, Portland, Oreg., 1987). The irones (violet-scented ketonic compounds) are expensive materials commonly used in cosmetics and perfumes (Gozu et al., Plant Cell Rpt., 13:12-16, 1993).
Iris germanica is one of the horticulturally most important tall bearded irises in the genus. Hundreds of valuable cultivars from this species have been developed and cultivated commercially as perennial ornamental plants. Traditionally, rhizomatous iris plants are propagated by splitting rhizomes, with a maximum annual yield of 10 plants/rhizome (Jxc3xa9han et al., Plant Cell Rep., 13:671-675, 1994). This practice is inefficient and slow, especially for propagating new cultivars for commercial use. Propagation by seed is impractical because of low germination rates and the allogamous nature of iris. Therefore, a more efficient propagation method is needed.
Plant regeneration from somatic tissues is generally considered a prerequisite to genetic transformation. Many efforts have been made to induce plant regeneration via in vitro callus culture of various explant types from several iris species (Fujino et al., J. Jpn. Soc. Hort. Sci., 41:66-71, 1972; Gozu et al., Plant Cell Rep., 13:12-16, 1993; Hussey, Scientia Hort., 4:163-165, 1976; Laublin et al., Plant Cell Tiss. Org. Cult., 27:15-21, 1991; Meyer, Jr., et al., HortScience, 10:479-480, 1975; Radojevixc4x87 and Landre, Proc. 7th Intern Congr. Plant Tissue and Cell Culture, Amsterdam, The Netherlands, (Abstr.) B4-100, 1990; Radojevixc4x87, et al. Acta Hort., 212:719-723, 1987; Radojevixc4x87 and Subotixc4x87, J. Plant Physiol., 139:690-696, 1992; van der Linde et al., Acta Hort. 226:121-128, 1988; Yabuya et al., Euphytica 57:77-81, 1991). In I. germanica, Reuther (Ber. Deutsch Bot. Ges., 90: 417-437, 1977) induced embryogenic calli from zygotic embryos and Jxc3xa9han et al. (Plant Cell Rep., 13:671-675, 1994) regenerated plants via somatic embryogenesis from leaves, rhizome apices, and immature flowers. Shimizu et al. cultured protoplasts and regenerated plants via somatic embryogenesis (Euphytica, 89:223-227, 1996). The same group induced embryogenic calli from three cultivars of I. germanica, but was able to induce regeneration from suspension cultures in only one (Shimizu et al., Plant Cell Tiss. Org Cult., 50:27-31, 1997). The low efficiency of plant regeneration in I. germanica and other iris species has hindered development of a suitable system for genetic transformation. Genetic transformation of iris has not previously been reported.
Strong consumer demand means increased challenges in developing new iris cultivars with novel characteristics. Unfortunately, most efforts in iris breeding have been primarily intraspecific because of the high degree of incompatibility between species. Thus, the search for an alternative breeding method is imperative. Genetic transformation and regeneration offers an alternative approach for introducing desirable traits, such as resistance to herbicides, diseases, and insects; or developing desired floral characteristics such as novel colors.
The inventors have developed efficient A. tumefaciens-mediated and microparticle bombardment transformation methods and regeneration methods for ornamental monocots such as Iris. With the provision herein of such transformation and regeneration methods, rapid and efficient iris transformation and/or in vitro propagation is now enabled.
Embodiments of the invention may include methods of transforming iris cells; such methods involve introducing a recombinant nucleic acid molecule into an iris cell, initiating callus formation from the iris cells; and selecting transformed cells. Selection of transformed cells can, for instance, involve growing the cells on medium that provides a selective pressure towards the transformed cells. The recombinant nucleic acid can be introduced in any manner, including co-cultivating the iris cells with Agrobacterium (e.g., A. tumefaciens or rhizogenes); bombarding the cells with nucleic acid-coated microprojectiles; and electroporating or PEG treatment of protoplasts of the cells.
In certain embodiments of the invention, the iris cells to which the recombinant nucleic acid is introduced are in suspension culture; however, they could also be in callus culture. Alternatively, these cells could be cells excised directly from an iris plant, such as meristematic cells or other partially differentiated or de-differentiated cells. Cells useful for transformation and/or regeneration as described herein include cells from iris shoot tissue, root tissue, rhizome tissue, and flower or other reproductive tissue.
Specific methods disclosed further include regenerating transformed shoots from the transformed plant cells. The disclosed methods may also include inducing root formation in the transformed cells and/or shoots.
Another embodiment of the invention includes a method for transforming iris cells, wherein the iris cells are co-cultivated with an Agrobacterium that contains a recombinant vector (e.g., a regular binary vector, a co-integrating vector, or a super binary vector). Such a recombinant vector can include a transfer DNA region, and may further include at least one (but often, more than one) protein-encoding sequence. Such protein-encoding sequences can include, for instance, selectable marker genes and/or desired trait genes (e.g., those encoding irone synthetic proteins, plant pigment synthetic proteins, pesticide resistance proteins, herbicide resistance proteins, or disease resistance proteins).
Methods of transformation and regeneration as disclosed herein find equal application in any species of the genus Iris, including Iris germanica, I. hollandica, I. pallida, I. setosa, I. lavigata, and I. pumila. Likewise, the described methods are effective independent of the ploidy of the Iris , and therefore find equal application in, for instance, diploid, tetraploid, and hexaploid varieties, as well as variants that are aneuploid for one or more chromosomes.
Also encompassed by this invention are cells produced by the disclosed transformation and/or regeneration methods, and plants, plant parts (including seeds and flowers), and plant progeny produced using such transformed and/or regenerated cells. In certain embodiments, these cells/tissues/plants will express one or more traits that the cell source material (source explant or explant) did not posses, such as an altered flower color, flowering time, disease resistance, herbicide resistance, pesticide resistance, or senescence schedule.
A further embodiment of the invention includes a method for culturing iris cells and regenerating iris plants, which includes growing suspension culture in MS-L medium supplemented with an auxin and a cytokinin (e.g., about 5 xcexcM 2,4-D and about 0.5 xcexcM Kin) in the dark for a period of time (for instance, six weeks) at 25xc2x0 C., and isolating relatively small cell clusters (e.g., those about xe2x89xa6520 xcexcm) from the suspension culture. This method can further include inoculating the isolated clusters into an appropriate shoot induction medium (e.g., MS-I medium supplemented with about 2.5 to about 12.5 xcexcM Kin and 0.0 to about 0.5 xcexcM NAA) and growing the clusters in the dark for another period of time (for instance, about six weeks) at 25xc2x0 C. to initiate differentiation. Differentiated clumps can then be isolated, and placed on shoot elongation and development medium (e.g., MS-D with 1.25 xcexcM DA) under light (for instance, about 50 xcexcm/m2s) at 23xc2x0 C. for a period sufficient to regenerate shoots and/or plantlets (which in some embodiments will be about six weeks). Regenerated shoots and/or plantlets can be transferred to root initiation medium, and subsequently transplanting rooted shoots and/or plantlets to soil in a greenhouse.
In a further embodiment of the invention, iris cells can be transformed with a recombinant nucleic acid molecule prior to being regenerated by this method. Such regeneration can for instance include co-cultivation with A. tumefaciens or microparticle bombardment.
The foregoing and other features and advantages of the invention will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures and tables.